Adenovirus vectors are an excellent gene transfer vector for animal cells because of the high efficiency of gene transfer, the ability of allowing a gene to be transferred into nondividing cells, the ease of preparing a high titer virus stock, and the like, and clinical applications thereof have been attempted as a vector for use in gene therapy. Adenovirus vectors that are widely used currently are replication-defective vectors in which the E1 gene essential for the replication of adenovirus and the expression of all adenovirus proteins has been deleted, and the vectors are termed as the first generation adenovirus vectors (some of them are further comprise the deletion of the E3 gene).
It was thought that since the first generation adenovirus vectors lack the E1 gene, none of adenovirus proteins could be expressed in normal cells such as human cells that are not expressing the E1 gene. However, recent studies have demonstrated that when a gene was transferred using a first generation adenovirus vector in vivo, expression of the transgene has been transient, and an inflammatory response occurs at the site of gene transfer (Yang Y. et al., Proc. Natl. Acad. Sci. USA 91: 4407-4411 (1994), and Wilmott R. W. et al., Hum. Gene Ther., 7: 301-318 (1996)).
As an explanation of the cause, the following hypothesis was proposed. In cells into which first generation adenovirus vectors have been infected, adenovirus early genes such as the E2A gene are expressed at low levels by transcription factors derived from cells, and thereby the adenovirus major late promoter (MLP) are activated. Subsequently, major late proteins are expressed, and cytotoxic T lymphocytes (CTL) against them are lead to generate, and CTL eliminates the foreign gene-transferred cells. Thus, it is a theory that the expression of adenovirus late proteins via the expression of adenovirus early proteins is a cause for the inflammatory response and the lack of persistent expression of the transgenes (Engelhardt J. F. et al., Proc. Natl. Acad. Sci. USA 91: 6196-6200 (1994), and Yang Y. et al., Nature Gnent., 7: 362-368 (1994)).
Based on this hypothesis, various improved adenovirus vectors were constructed in which genes essential for the replication of adenovirus other than the E1 gene were deleted, and then supplied with the proteins encoded by the genes from virus producing cells, the virus was made possible to replicate. For example, there have been reported an adenovirus vector in which the E2A gene has been deleted (Zhou H. et al., J. Virol., 70: 7030-7038 (1996), and Gorziglia M. I. et al., J. Virol., 70: 4173-4178 (1996)), an adenovirus vector in which the E4 gene which is an early gene as is the E2A gene has been deleted (Krouglicak V. et al., Hum. Gene Ther., 6: 1575-1586 (1995), and Wang Q. et al., Gene Ther., 2: 775-783 (1995), Yeh P. et al., J. Virol., 70: 559-565 (1996)), and the like. However, for adenovirus vectors in which the E2A gene has been deleted, little effect has been observed of prolonging the expression period of the transgenes and of reducing inflammatory responses (Morral N. et al., Hum. Gene Ther., 8: 1275-1286 (1997), and Lusky M. et al., J. Virol., 72: 2022-2032 (1998), and O'neal W. K. et al., Hum. Gene Ther., 9: 1587-1598 (1998)). Furthermore, it has been reported that in an adenovirus in which the E4 gene has been deleted, a moderate improvement was observed but was not satisfactory (Gao G-P. et al., J. Virol., 70: 8934-8943 (1996), Wang Q. et al., Gene Ther., 4: 393-400 (1997), and Dedieu J-F. et al., J. Virol., 71: 4626-4637 (1997)).
Thus, as a further improved vector, a helper-dependent adenovirus vector (HD vector) was proposed, in which all adenovirus genes were deleted except the inverted terminal repeat (ITR) and the packaging signal and which replicate depending on a helper virus (Kochanek S. et al., Proc. Natl. Acad. Sci. USA 93: 5731-5746 (1996), and Parks R. J. et al., Proc. Natl. Acad. Sci. USA 93: 13565-13570 (1996)). This HD vector is also termed as a gutted vector or a gutless vector. Said HD vector has been reported to exhibit an effect of improved vector such as the extension of the expression period of the transgene and reduction in inflammatory responses (Morsy M. A. et al., Proc. Natl. Acad. Sci. USA 95: 7866-7871 (1998), Schiedner G. et al., Nature Gnent. 18: 180-183 (1998), and Morral N. et al., Hum. Gene Ther. 9: 2709-2716 (1998)).
However, when this HD vector is clinically used as a pharmaceutical drug, there is a big problem of low productivity of vectors. The reason is as follows: First, in the production of HD vectors, HD vectors depend on helper adenoviruses for the supply of all proteins required for the replication as their source. From the principle of production to enable the replication of HD vectors while always maintaining the helper viruses at a constant ratio, the reported yield of HD vectors produced per cells is evidently low compared to the first generation adenovirus vectors (Morsy M. A. et al., Proc. Natl. Acad. Sci. USA 95: 7866-7871 (1998), Schiedner G. et al., Nature Gnent. 18: 180-183 (1998)). Furthermore, since the desired HD vector is always contaminated with the helper virus used for the production, the helper virus must be removed by separating the virus by ultracentrifugation based on a subtle difference in specific gravity between the viruses. Sometimes the first generation adenovirus vectors are purified by ultracentrifugation, but its purpose is to remove contaminating proteins from adenovirus particles. On the other hand, in the purification of HD vectors, not only the contaminating proteins must be removed but, as described above, helper viruses must be separated, and therefore the yield to be purified at one run of centrifugation is smaller than for the first generation adenovirus vectors. If helper viruses have not been adequately removed, the safety of viruses per se remains a concern. Furthermore, though the first generation adenovirus vectors can also be purified by the column method (Huyghe B. D. et al., Hum. Gene Ther. 6: 1403-1416 (1995)) that can handle a larger quantity of viruses than by ultracentrifugation method, the column method cannot be applied by the present state of art due to the necessity of removal of helper viruses for HD vectors. For the above reasons, the productivity of HD vectors is clearly lower than that of the first generation adenovirus vectors in both the production from cultured cells and in the subsequent purification steps. Therefore, to produce HD vectors in an amount sufficient for clinical applications is considered to be extremely difficult and is impractical.
Furthermore, since virus-derived promoters such as cytomegalovirus (CMV) promoters and Rous sarcoma virus (RSV) promoters have been widely used for vectors for gene therapy including adenovirus vectors because their promoter activities are high levels. However, it has been reported that in order to attain a long-term expression in vivo of the desired gene from these promoters, protein encoded by the adenovirus E4 gene is required (Armentano D. et al., J. Virol., 71: 2408-2416 (1997), and Brough D. E. et al., J. Virol. 71: 9206-9213 (1997)). From the standpoint of persistent promoter activity, adenovirus vectors that have inserted therein high-activity promoters derived from viruses must use vectors having a structure similar to those of the first generation adenovirus vectors.